This invention relates to novel polypeptide and protein derivatives in which polypeptides and proteins are conjugated by bridging molecules to the same kind of polypeptides or proteins, other kinds of proteins or polypeptides, reporter groups or cytotoxic agents.
In the diagnosis of many forms of disease, as well as when following the effects of treatment, it would often be desirable to use labelled proteins that bind to specific target structures in the body. For example, when diagnosing or treating cancer, it would be desirable to be able to detect both primary tumours and metastases using labelled tumour-specific antibodies. Many reports have appeared on the labelling of proteins and antibodies by random chemical attack on their side chains. In such a process, most frequently, the side chains of the tyrosines are iodinated (Mach et al., Cancer Research 43, 5593-5600 [1983]), or the side-chains of the lysines are acylated. In this latter case the acylation is often by groups that chelate metals (e.g. Hnatowich et al., Science 220, 613-615 [1983]). Subsequently, the chelating groups can be used to bind radioactive metals. It has also been suggested but not yet been satisfactorily tested to bind to such molecules paramagnetic ions for nuclear magnetic resonance (NMR) imaging (Brady et al., Magnetic Resonance in Medicine 1, 286 [1984]). The labelling of proteins, especially of antibodies, however, has so far always been effected in a more or less random way.
Random substitutions on biological active proteins, for example random substitutions on antibody molecules, can have a number of drawbacks:
1. If by chance a particularly reactive site were to lie in the active site of the protein a substitution at this site would possibly inactivate the protein, e.g. a particularly valuable monoclonal antibody might be rendered totally useless if by chance a side chain particularly reactive towards substitution were to lie in the antigen-binding site. The substitution would then inactivate the antibody.
2. Even when the active site of the protein (e.g. an antibody) escapes serious damage, a high number of substitutions on the proteinxe2x80x94which may be desirable, e.g. in order to have a high intensity in case of radioactive labelling via chelating groupsxe2x80x94might change its physico-chemical properties (e.g. solubility).
3. A random, multiple substituted product constitutes a heterogenous mixture of molecules with different properties, with attendant problems of assuring constant properties from batch to batch.
The present invention relates to novel polypeptide and protein derivatives, to a process for their preparation, to their use and to novel intermediates therefor. The novel polypeptides and proteins of the present invention are, more specifically, polypeptides and proteins which are conjugated via an intermediate grouping containing at least one radical of the formula xe2x80x94C(R)xe2x95x90Nxe2x80x94 (or xe2x80x94Nxe2x95x90C(R)xe2x80x94) or xe2x80x94CH(R)xe2x80x94NHxe2x80x94 (or xe2x80x94NHxe2x80x94CH(R)xe2x80x94), wherein R is hydrogen, an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group which group may be substituted, with themselves or each other, with a different polypeptide or protein or with a reporter group or a cytotoxic agent. These compounds are obtained by condensation of two reactants one of which is an aldehyde (or acetalized aldehyde) or ketone the other being an amino compound thus yielding a Schiff base or azomethine type compound which, if desired or necessary, can be stabilized in a further reaction, viz. by reduction of the xe2x80x94C(R)xe2x95x90Nxe2x80x94 (or xe2x80x94Nxe2x95x90C(R)xe2x80x94) radical to a xe2x80x94CH(R)xe2x80x94NHxe2x80x94 (or xe2x80x94NHxe2x80x94CH(R)xe2x80x94) radical.
The present invention in a major aspect makes use of the fact that enzymes can direct bifunctional reagents with suitable reactive groups at specific sites in polypeptides or proteins (e.g. antibodies). These sites are preferably the carboxyl terminus of the polypeptide chain, which is at least in terms of primary structure in most cases far from the active site of proteins. This is especially true for antibody molecules where the carboxyl terminus is furthest away from the antigen-binding site. Therefore problem No. 1 mentioned above can be eliminated by the process of the present invention. The limitation of the substitution to a specific site such as the carboxyl terminus, will also eliminate problems No. 2 and No. 3, above.
However, in a further aspect the present invention makes use of the fact that specific bifunctional reagents with suitable reactive groups preferably or specifically react at non carboxy terminus sites of the molecule, viz. with specific side chains or the amino terminal amino group in a non-enzymatic reaction.
Examples of bifunctional reagents with suitable reactive groups are compounds with an amino group at one end and with a formyl or amino group (preferably in protected form) at the other end, such as o-, m- or p-formylphenylalanine.
Therefore, the polypeptide and protein derivatives of the present invention can be prepared by a condensation reaction between an aldehyde or ketone and an amino compound to yield the desired derivative of the azomethine or Schiff base type and, if desired, subsequent reduction of the xe2x80x94Cxe2x95x90Nxe2x80x94 radical (which is relatively labile in case one of the reaction partners is an amine and the product is a Schiff base) to form a corresponding derivative containing a xe2x80x94CH2xe2x80x94NHxe2x80x94 radical. The amino compound can be an amine, an O-alkylated hydroxylamine or a hydrazide. In the case of an O-alkylated hydroxylamine reacting with a carbonyl compound (aldehyde or ketone) oximes are obtained containing a xe2x80x94C(R)xe2x95x90Nxe2x80x94Oxe2x80x94 radical. Since such compounds are relatively stable no subsequent reduction, albeit possible, is necessary to form a corresponding derivative containing a xe2x80x94CH(R)xe2x80x94NHxe2x80x94Oxe2x80x94 radical. In the case of a hydrazide reacting with a carbonyl compound the reaction product will contain a xe2x80x94C(R)xe2x95x90Nxe2x80x94NHxe2x80x94 radical which again is relatively stable and needs no reduction to form a corresponding derivative containing a xe2x80x94CH(R)xe2x80x94NHxe2x80x94NHxe2x80x94 radical.
The basic reaction scheme of which the present makes use is  greater than Cxe2x95x90O+H2Nxe2x80x94xe2x86x92 greater than Cxe2x95x90Nxe2x80x94xe2x86x92 greater than CHxe2x80x94NHxe2x80x94. In this scheme, one complementary group (carbonyl or amino) is placed at the N- or C-terminus of a protein or polypeptide under mild conditions. To obtain specificity (discrimination between an attached amino group and lysine side chains of the protein or polypeptide) a reactive amino group attached to a protein must be an aromatic one, i.e. must be directly attached to an aromatic group, such as phenyl or it must be directly attached to xe2x80x94Oxe2x80x94 or to xe2x80x94NHxe2x80x94COxe2x80x94, i.e. be an O-alkyl-hydroxylamine or a hydrazide, respectively.
If at least one of the reactive groups (carbonyl or amino group) of the reaction partners is aromatic, preferably if both are aromatic, it was found that the condensation reaction is rapid, and highly efficient even at surprisingly low concentrations of reactants. The reactivities involved are sufficiently great to permit the attachment of, e.g. a polymeric chelating group to the specific site, which means that at the cost of a single modification at a specific site on the protein known to be safe for this purpose, it is possible to introduce virtually as many of the desired substituent groups as required for high radioactivity. This feature again permits to overcome problem No. 2, addressed above, since a high number of substitutions spread over the whole protein in order to achieve a high enough intensity of labelling is no longer required.
For the reasons discussed above, it is usually preferable to have the group which is to participate in the condensation reaction to form a Schiff base type compound attached specifically, via enzymatic methods, to the carboxy terminus of the protein or polypeptide.
Under certain circumstances, however, it may be satisfactory and convenient to form Schiff base links via groups introduced elsewhere and groups introduced by other methods. Usually, but not always, such methods are less specific than the carboxy-terminal enzymatic method. Circumstances under which these other methods might be employed are:
(i) in cases where the carboxy-terminal region is important for function or should not be altered for other reasons and
(ii) where particular properties of the protein or polypeptide, e.g. its possessing a single or rather few side chain residues of an amino acid for which specific chemical modification reagents exist or may be designed, can usefully be exploited.
Therefore, it is possible to combine group-specific chemical modification of protein or polypeptide side chains with subsequent coupling to Schiff bases. In the context of the present specification and claims the term xe2x80x9cSchiff basexe2x80x9d is meant to extend to all protein or polypeptide derivatives exhibiting a  greater than Cxe2x95x90Nxe2x80x94 radical and thus also encompasses compounds, such as, oximes or hydrazones. A wide variety of group-specific protein modification reagents are known which permit the modification, with various degrees of specificity, of the functional groups present in proteins. Furthermore, many examples exist where two of the chemically reactive groups present in such reagents have been incorporated into a single molecule to provide a bivalent reagent (see e.g. the Catalogue of the Pierce Chemical Co., the world""s leading manufacturer of protein cross-linking reagents). So far, none of these reagents have been used for Schiff base chemistry. It should be noted that great advantage is to be made from combining, in the same molecule, a group capable of reacting with functional groups of proteins or polypeptides and a group capable (after deprotection, if it is used in protected form) of forming a Schiff base link with a complementary group on another molecule, viz. a carbonyl or amino group. Such reagents are represented by the general formula R3xe2x80x94Xxe2x80x94R1, where R3 is a chemical group which reacts with functional groups of proteins or polypeptides, X is a bivalent organic group or may be absent but is preferably an aromatic radical directly adjacent to R1 and must be an aromatic group or oxygen directly adjacent to R1 where R1 is amino or protected amino and R1 is carbonyl, acetalised formyl (e.g. dimethoxy or diethoxy methyl), amino or protected amino. Suitable amino protecting groups are those which are stable enough to withstand the attachment of R3 to the polypeptide or protein, yet labile enough to be removed under conditions which do not irreversibly denature the polypeptide or protein. Many such groups are known to the art, e.g., citraconyl, trifluoroacetyl, Boc, BPOC, MSC. Suitable groups for R3 are well known to the art (c.f., for example, Means, G. E. and Feeney, R. E. [1971] xe2x80x9cChemical Modification of Proteinsxe2x80x9d, Holden-Day, San Francisco): groups that react selectively with amino-groups are, e.g., active-esters such as hydroxysuccinimide esters, o-nitrophenyl esters, imidates or haloaromatics with a nucleus activated to nucleophilic substitution; groups that react selectively with sulphhydryl-groups are, e.g., haloalkyls, activated disulphides, aziridines, activated vinyl compounds; groups that react selectively with guanido-groups are, e.g., alpha- or beta-dicarbonyls; aromatic-group selective reagents are, e.g., diazonium compounds; indole-group selective compounds are, e.g., aromatic sulphenyl halides, and carboxyl-group selective reagents are, e.g., diazoalkanes and amino compounds in the presence of condensing reagents such as DCCI.
The polypeptide and protein derivatives of the present invention can be represented by the formula
Axe2x80x94Xxe2x80x94Zxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I)
wherein
A is the residue of a protein or polypeptide;
B is the residue of a protein or polypeptide, of a reporter group or of a cytotoxic agent;
X and Xxe2x80x2 independently from each other are bivalent organic radicals or may be absent;
Z is a bivalent radical selected from the group consisting of xe2x80x94C(R)xe2x95x90Nxe2x80x94, xe2x80x94Nxe2x95x90C(R)xe2x80x94, xe2x80x94CH(R)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94CH(R)xe2x80x94, xe2x80x94C(R)xe2x95x90Nxe2x80x94Yxe2x80x94Nxe2x95x90C(R)xe2x80x94, xe2x80x94Nxe2x95x90C(R)xe2x80x94Yxe2x80x94C(R)xe2x95x90Nxe2x80x94, xe2x80x94CH(R)xe2x80x94NHxe2x80x94Yxe2x80x94NHxe2x80x94CH(R)xe2x80x94 or xe2x80x94NHxe2x80x94CH(R)xe2x80x94Yxe2x80x94CH(R)xe2x80x94NHxe2x80x94, wherein R is hydrogen, an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group, which group may be substituted with the same or a different protein or polypeptide, a reporter group or a cytotoxic agent, with at least one aromatic radical or oxygen adjacent to nitrogen and
Y is a bivalent organic group
and salts thereof.
The invention also extends to salts of the protein and polypeptide derivatives mentioned above, especially to metal salts thereof. Of most interest among the salts are the metal chelate complexes useful for in vivo imaging.
Thus, the compounds of formula I are derivatives of (or modified) polypeptides or proteins. Typical polypeptides and proteins, the residues of which are designated A and B in formula I, are on the one hand those occurring in nature and capable of being isolated from nature independent from whether their structure and/or amino acid sequences and glycosylation pattern has already been identified or not and on the other hand those which have been or can be prepared synthetically or semisynthetically in accordance with methods well-known in the art. Preferred compounds of formula I are derivatives of polypeptides and proteins of medical interest, among which, e.g., derivatives of immunoglobulins, especially antibodies of the IgG type. It will be appreciated that not only complete antibodies can be labelled or derivatised using the method of the present invention but also subunits thereof which are still functional, such as F(abxe2x80x2)2 or Fab fragments.
Formula I comprises compounds of the following types:
Axe2x80x94Xxe2x80x94C(R)xe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Axe2x80x2)
Axe2x80x94Xxe2x80x94Nxe2x95x90C(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Axe2x80x3)
Axe2x80x94Xxe2x80x94CH(R)xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Bxe2x80x2)
Axe2x80x94Xxe2x80x94NHxe2x80x94CH(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Bxe2x80x3)
Axe2x80x94Xxe2x80x94C(R)xe2x95x90Nxe2x80x94Yxe2x80x94Nxe2x95x90C(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Cxe2x80x2)
Axe2x80x94Xxe2x80x94Nxe2x95x90C(R)xe2x80x94Yxe2x80x94C(R)xe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Cxe2x80x3)
Axe2x80x94Xxe2x80x94CH(R)xe2x80x94NHxe2x80x94Yxe2x80x94NHxe2x80x94CH(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Dxe2x80x2)
Axe2x80x94Xxe2x80x94NHxe2x80x94CH(R)xe2x80x94Yxe2x80x94CH(R)xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Dxe2x80x3)
wherein A, B, X, Xxe2x80x2, R and Y are as defined above.
With R=hydrogen (i.e. with one of the reaction partners being an aldehyde or a protected aldehyde) compounds of the following general formulae are obtained:
Axe2x80x94Xxe2x80x94CHxe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-axe2x80x2)
Axe2x80x94Xxe2x80x94Nxe2x95x90CHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-axe2x80x3)
Axe2x80x94Xxe2x80x94CH2xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-bxe2x80x2)
Axe2x80x94Xxe2x80x94NHxe2x80x94CH2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-bxe2x80x3)
xe2x80x83Axe2x80x94Xxe2x80x94CHxe2x95x90Nxe2x80x94Yxe2x80x94Nxe2x95x90CHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-cxe2x80x2)
Axe2x80x94Xxe2x80x94Nxe2x95x90CHxe2x80x94Yxe2x80x94CHxe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-cxe2x80x3)
Axe2x80x94Xxe2x80x94CH2xe2x80x94NHxe2x80x94Yxe2x80x94NHxe2x80x94CH2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-dxe2x80x2)
Axe2x80x94Xxe2x80x94NHxe2x80x94CH2xe2x80x94Yxe2x80x94CH2xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-dxe2x80x3)
wherein A, B, X, Xxe2x80x2 and Y are as defined above.
In the case where B represents a protein or polypeptide residue these residues may be different from or identical with A. In the first case hetero-dimers of proteins and polypeptides and in the second case homo-dimers of proteins and polypeptides can be obtained.
B may alternatively represent the residue of a cytotoxic agent or a reporter group. Cytotoxic agents in the present context are defined to comprise all compounds generally summarized under this expression such as cytostatics and toxins. Cytostatics of specific interest are those chemo-therapeutically active compounds against cancer, i.e. cancerostatics (carcinostatics). The term xe2x80x9creporter groupxe2x80x9d is meant to define compounds which are easily detectable by analytical means in vitro and/or in vivo and which confer this property to compounds to which they are bound. This term comprises, e.g., any organic compounds/groups which are capable of binding strongly to metals (including and preferably radioactive metals). Especially preferred among such reporter groups are metal chelating agents/groups (chelons), e.g., desferrioxamine or DTPA (systematic names see below). Apart from being a compound/group capable of being radioactively labelled, reporter groups may be fluorescent groups or groups capable of being monitored by NMR or ESR spectroscopy.
The groups X and Xxe2x80x2 can be absent or represent bivalent radicals of aliphatic, aromatic or araliphatic compounds and can be substituted. X and Xxe2x80x2 may be identical or can differ from each other, e.g., only one may be present. Preferred groups X and Xxe2x80x2 are aromatic radicals, e.g., xe2x80x94NHxe2x80x94C6H4xe2x80x94 or araliphatic radicals, e.g., xe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94C6H4xe2x80x94, xe2x80x94NHxe2x80x94CH(COOCH3)xe2x80x94CH2xe2x80x94C6H4xe2x80x94 or xe2x80x94NHxe2x80x94CH(CONH2)xe2x80x94CH2xe2x80x94C6H4xe2x80x94, since the forming of the Schiff base type compounds is favoured in this case. Examples of aliphatic groups X or Xxe2x80x2 are xe2x80x94Oxe2x80x94CH2xe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94. It is essential that in case there are aliphatic amino groups present in the protein or polypeptide molecule (which latter case generally happens, e.g., if lysine is present) that the aromatic group is adjacent to the N-atom of group Z of formula xe2x80x94Nxe2x95x90CHxe2x80x94 or xe2x80x94NHxe2x80x94CH2xe2x80x94, which means that the Schiff base formation occurs via an aromatic amino group at the side of the protein or polypeptide. In case the protein, polypeptide or the cytotoxic agent or reporter group contains already such an aromatic amino group through which the coupling can be effected X and/or Xxe2x80x2 will be absent. In this case the N-atom of Z originates from the starting protein, polypeptide, cytotoxic agent or the reporter compound. The same applies with respect to an aromatic formyl function.
In order to reach highest specificity in the coupling reaction it is preferred either to use aromatic aldehydes and aromatic amino compounds so that in the Schiff base compounds obtained the radicals adjacent to both the N- and the C-atom of the xe2x80x94CHxe2x95x90Nxe2x80x94 or xe2x80x94Nxe2x95x90CHxe2x80x94 group are aromatic groups, preferably phenylene groups, or to use ketones and O-alkylhydroxylamines.
In case one of the reaction partners is a ketone it is preferable to use amino compounds which are stronger nucleophiles than arylamino compounds and which are known to react rapidly, specifically and under mild conditions with carbonyl groups. Such amino compounds include substituted hydrazines (hydrazides) and O-substituted, preferably O-alkylated, hydroxylamines, such as H2Nxe2x80x94Oxe2x80x94CH2xe2x80x94COOH. The stability at non-extreme pH of hydrazones and oximes means that the reduction step  greater than Cxe2x95x90Nxe2x80x94Xxe2x86x92 greater than CHxe2x80x94NHxe2x80x94X, which is required when X is aryl, is not necessary, albeit possible. In the case of a hydroxylamino compound being used as nucleophile compound of formula I will be obtained wherein Z is a bivalent radical selected from the group consisting of xe2x80x94CHxe2x95x90Nxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94Nxe2x95x90C(R)xe2x80x94, xe2x80x94CH(R)xe2x80x94NHxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94NHxe2x80x94CH(R)xe2x80x94, xe2x80x94C(R)xe2x95x90Nxe2x80x94Oxe2x80x94Yxe2x80x94Oxe2x80x94Nxe2x95x90C(R)xe2x80x94, xe2x80x94Oxe2x80x94Nxe2x95x90C(R)xe2x80x94Yxe2x80x94C(R)xe2x95x90Nxe2x80x94Oxe2x80x94, xe2x80x94CH(R)xe2x80x94NHxe2x80x94Oxe2x80x94Yxe2x80x94Oxe2x80x94NHxe2x80x94CH(R)xe2x80x94 and xe2x80x94Oxe2x80x94NHxe2x80x94CH(R)xe2x80x94Yxe2x80x94CH(R)xe2x80x94NHxe2x80x94Oxe2x80x94, with R and Y being as defined above.
Compounds of formulae I-Cxe2x80x2, I-Cxe2x80x3, I-Dxe2x80x2 and I-Dxe2x80x3 are obtained when a diamino compound of formula H2Nxe2x80x94Yxe2x80x94NH2 or a dicarbonyl compound of formula OC(R)xe2x80x94Yxe2x80x94C(R)O is reacted with a carbonyl or an amino compound respectively. Y can be any bivalent organic group, i.e. an aliphatic, aromatic or araliphatic group. For obvious reasons simple molecules are preferred. A most preferred aromatic Y group is phenylene while in case of an aliphatic Y group this group has two O- or NH-radicals. It is also evident that although compounds of formulae I-Cxe2x80x2, I-Cxe2x80x3, I-Dxe2x80x2 and I-Dxe2x80x3 can be prepared using methods well-known in the art wherein A and B and/or X and Xxe2x80x2 are different, the preferred compounds of that type are those wherein B is identical with A and Xxe2x80x2 is identical with X (including the possibility that both latter groups are absent). Thus symmetric proteins or polypeptide dimers are obtainable which are coupled almost specifically via a xe2x80x94Cxe2x80x94Nxe2x80x94Yxe2x80x94Nxe2x80x94Cxe2x80x94 or a xe2x80x94Nxe2x80x94Cxe2x80x94Yxe2x80x94Cxe2x80x94Nxe2x80x94 chain.
The compounds of formula I and their salts in accordance with the process of the present invention are obtained by condensing a compound of formula
Axe2x80x94Xxe2x80x94R1xe2x80x83xe2x80x83(II)
wherein
R1 is xe2x80x94COxe2x80x94R, acetalized formyl or amino and
A is a residue of a protein of polypeptide, X is a bivalent organic radical or may be absent, and R is hydrogen or an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group, which group may be substituted with the same or a different protein or polypeptide, a reporter group or a cytotoxic agent,
with a compound of formula
R2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(III)
wherein
R2 is amino in case R1 in the compound II above is xe2x80x94COxe2x80x94R or acetalized formyl and is xe2x80x94COxe2x80x94R or acetalized formyl in case R1 in compound II above is amino and Xxe2x80x2 is a bivalent organic radical or may be absent, B is a residue of a protein or polypeptide, a reporter group or a cytotoxic agent, and R is hydrogen or an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group, which group may be substituted with the same or a different protein or polypeptide, a reporter group or a cytotoxic agent,
or condensing a compound of formula II above with a compound of formula
R2xe2x80x94Yxe2x80x94R2xe2x80x83xe2x80x83(IV)
wherein
Y is as defined above and
R2 is amino in case R1 in the compound II above is xe2x80x94COxe2x80x94R or acetalized formyl and is xe2x80x94COxe2x80x94R or acetalized formyl in case R1 in compound II above is amino to form a Schiff base and, if desired, reducing the xe2x80x94C(R)xe2x95x90Nxe2x80x94 or xe2x80x94Nxe2x95x90C(R)-radical(s) generated by the condensation to xe2x80x94CH(R)xe2x80x94NHxe2x80x94 or xe2x80x94NHxe2x80x94CH(R)-radical(s) respectively and, if desired, forming a salt.
Thus either a carbonyl compound Axe2x80x94Xxe2x80x94C(R)O, in case of Rxe2x95x90H an aldehyde or an acetal thereof, preferably the methyl or ethyl acetal, is reacted with an amino compound, preferably an aromatic amine H2Nxe2x80x94Xxe2x80x2xe2x80x94B or an O-derivative of hydroxylamine, or an amino compound, preferably an aromatic amine Axe2x80x94Xxe2x80x94NH2 or an O-derivative of hydroxylamine, is reacted with a carbonyl compound O(R)Cxe2x80x94Xxe2x80x2xe2x80x94B, in case of Rxe2x95x90H an aldehyde or a corresponding acetal, preferably the methyl or ethyl acetal, to form the Schiff base.
If symmetric bisproteins or bispolypeptides are desired a carbonyl compound Axe2x80x94Xxe2x80x94C(R)O or an acetal thereof, in case Rxe2x95x90H, is reacted with a diamino compound H2Nxe2x80x94Yxe2x80x94NH2, or an amino compound Axe2x80x94Xxe2x80x94NH2, preferably an aromatic amino compound or an O-derivative of hydroxylamine, is reacted with a carbonyl compound O(R)Cxe2x80x94Yxe2x80x94C(R)O or an acetal thereof, in case Rxe2x95x90H.
As follows from the definitions of A, B and Z above the amino or carbonyl (or acetalized formyl) groups R1 and R2 in compounds of formulae II and III which participate in the formation of the Schiff base type bond are connected to the residues A and/or B either via the bivalent organic group X and/or Xxe2x80x2 respectively or may be part of the residues A and B respectively, in which latter case X and/or Xxe2x80x2 in the resulting compound of formula I are/is absent.
It should be noted that at least one of the reacting carbonyl and amino groups is an aromatic group, viz. is directly connected to an aromatic group so that in a compound of formula I at least one aromatic group is directly adjacent to Z or that, alternatively, in case of an aliphatic carbonyl compound the amino compound is a hydrazide or hydroxylamino O-derivative.
Consequently, if in a compound Axe2x80x94Xxe2x80x94R1 (II) X has an aliphatic group adjacent to R1, the reactive carbonyl or amino function R2 in the compound of formula III must be an aromatic or araliphatic one, i.e. Xxe2x80x2 must have an aromatic group adjacent to R2 or the amino function should be derived from hydrazine or hydroxylamine, while if X has an aromatic group adjacent to R1 or the amino function is derived from hydrazine or hydroxylamine, the reactive carbonyl or amino function, R2 in the compound of formula III may be adjacent either to an aromatic or aliphatic group, but aromatic is preferred.
The structure of the aliphatic or aromatic groups Xxe2x80x2 and/or X is not critical. The aromatic groups may be derived from a hydrocarbon or from a heterocycle; they are preferably derived from benzene, viz. either of them is or both are phenylene radicals which may be substituted. The only limitation with respect to the substituents of Xxe2x80x2 and/or X is that they should not interfere with the reaction of the amino or carbonyl group, i.e. should not react instead of the amino or carbonyl groups, should not be a sterical hindrance or should not deactivate the reactive groups.
Compounds of the following general formulae are examples of subgroups of compounds of the general formula I
Axe2x80x94C(R)xe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Exe2x80x2)
Axe2x80x94Xxe2x80x94C(R)xe2x95x90Nxe2x80x94Bxe2x80x83xe2x80x83(I-Fxe2x80x2)
Axe2x80x94C(R)xe2x95x90Nxe2x80x94Bxe2x80x83xe2x80x83(I-Gxe2x80x2)
Axe2x80x94Nxe2x95x90C(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Exe2x80x3)
xe2x80x83Axe2x80x94Xxe2x80x94Nxe2x95x90C(R)xe2x80x94Bxe2x80x83xe2x80x83(I-Fxe2x80x3)
Axe2x80x94Nxe2x95x90C(R)xe2x80x94Bxe2x80x83xe2x80x83(I-Gxe2x80x3)
Axe2x80x94C(R)xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Hxe2x80x2)
Axe2x80x94Xxe2x80x94C(R)xe2x80x94NHxe2x80x94Bxe2x80x83xe2x80x83(I-Ixe2x80x2)
Axe2x80x94C(R)xe2x80x94NHxe2x80x94Bxe2x80x83xe2x80x83(I-Kxe2x80x2)
Axe2x80x94NHxe2x80x94C(R)xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-Hxe2x80x3)
Axe2x80x94Xxe2x80x94NHxe2x80x94CH(R)xe2x80x94Bxe2x80x83xe2x80x83(I-Ixe2x80x3)
and
Axe2x80x94NHxe2x80x94CH(R)xe2x80x94Bxe2x80x83xe2x80x83(I-Kxe2x80x3).
With R=hydrogen (i.e. with one of the reaction partners being an aldehyde or a protected aldehyde) compounds of the following subgroups of general formula I are obtained:
Axe2x80x94CHxe2x95x90Nxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-exe2x80x2)
Axe2x80x94Xxe2x80x94CHxe2x95x90Nxe2x80x94Bxe2x80x83xe2x80x83(I-fxe2x80x2)
Axe2x80x94CHxe2x95x90Nxe2x80x94Bxe2x80x83xe2x80x83(I-gxe2x80x2)
Axe2x80x94Nxe2x95x90CHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-exe2x80x3)
Axe2x80x94Xxe2x80x94Nxe2x95x90CHxe2x80x94Bxe2x80x83xe2x80x83(I-fxe2x80x3)
Axe2x80x94Nxe2x95x90CHxe2x80x94Bxe2x80x83xe2x80x83(I-gxe2x80x3)
Axe2x80x94CH2xe2x80x94NHxe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-hxe2x80x2)
Axe2x80x94Xxe2x80x94CHxe2x80x94NHxe2x80x94Bxe2x80x83xe2x80x83(I-ixe2x80x2)
xe2x80x83Axe2x80x94CH2xe2x80x94NHxe2x80x94Bxe2x80x83xe2x80x83(I-kxe2x80x2)
Axe2x80x94NHxe2x80x94CH2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x83xe2x80x83(I-hxe2x80x3)
Axe2x80x94Xxe2x80x94NHxe2x80x94CHxe2x80x94Bxe2x80x83xe2x80x83(I-ixe2x80x3)
and
Axe2x80x94NHxe2x80x94CH2xe2x80x94Bxe2x80x83xe2x80x83(I-kxe2x80x3).
The condensation between compounds II and III can be carried out in accordance with methods well-known in the art under mild conditions in dilute solutions. The reaction the most intensively studied was that of des-AlaB30-insulin-B29-formylanilide with m-aminobenzoyl-ferrioxamine B. The most generally useful ranges of conditions are described immediately below. However, as will be seen from the appended Examples the reaction conditions are easily and successfully applicable to other reactants in spite of considerable differences in their nature.
The reaction can be carried out with good results at a pH range of 3.5-5.5. Any suitable aqueous buffer can be used. The buffer was normally aqueous acetic acid (1%, v/v) adjusted to the desired value with NaOH solution. Insulin derivatives are poorly soluble at the upper end of this pH range, but the addition of solid urea overcame this problem without any detectable effect on coupling efficiency. Dimethylformamide could also be used as a solubilizing agent, at the cost of some slowing down of the coupling. Similar problems with other protein derivatives may be overcome in a similar way.
The concentration of the insulin-aldehyde derivative was usually between 500 xcexcM and 1 mM, that of the other reactant, m-aminobenzoyl ferrioxamine B, was usually between 500 xcexcM and 2.5 mM. The reactants can be used in equimolar amounts or up to a multiple excess of one of the components.
Couplings can be carried out at ambient temperature. The half-time of these reactions, as judged by HPLC after quenching by dilution in acid, is of the order of one to two minutes. After 20-40 minutes yields are generally already at a maximum and the starting product is almost imperceptible.
The protein-carbonyl derivative can be used either as the free carbonyl compound or, especially in case of aromatic aldehyde, as an acetal, preferably as the methyl or ethyl acetal. The free compound still coupled efficiently after storage as a freeze-dried powder at room temperature for some months. In theory, the acetal-protected forms should have been deprotected before coupling, but it proved possible to take advantage of their lability to acid below pH 6 (which is far greater in case of aromatic acetals, than the lability of aliphatic acetals) and allow them to deprotect in the coupling mixture. If it turns out that at pH 5.5 no coupling occurs, the pH may be lowered. At pH 3.5 there will most probably be no difference in the reaction speed between the acetal protected form and the free aldehyde.
When of appropriate structure the compounds of the Schiff base-type obtained may be isolated and purified. It is well-known that Schiff bases are readily hydrolyzed and relatively unstable due to easy cleavage of the xe2x80x94CHxe2x95x90N-bond. However, in some cases such lability may be of advantage and, therefore, explicitly desired. Schiff base-type compounds obtained from two aromatic reactants (aromatic aldehyde group and aromatic amino group) are more stable than those obtained with one of the reactants being aliphatic. Oximes and hydrazones are more stable than simple Schiff bases.
Therefore, it is generally desirable to stabilize the Schiff base-type compounds. This is most conveniently done by reduction of the xe2x80x94CHxe2x95x90N-bond to a xe2x80x94CH2xe2x80x94NH-bond and accomplished in a manner well-known in the art using complex metal hydrides, preferably sodium cyanoborohydride or pyridine borane. Only a small excess of cyanoborohydride is necessary and technical grade product can be used without disadvantage. Where a higher purity is desirable it can be purified by precipitation from acetonitrile by the method of Jentoft and Dearborn (J. Biol. Chem. 254, 4359-4365 [1979]). Otherwise, pyridine borane may be used (Wong et al., Anal. Biochem. 139, 58-67 [1984]).
The compounds of formula I (conjugates) obtained can easily be transformed into salts using methods well known in the art. In the case of conjugates wherein B is the residue of a chelating agent (chelon), metal salts, especially salts with radioactive metals are the desired end products useful as valuable tools in diagnosis and therapy. Every kind of radioactive salt can be obtained by simply mixing a protein-chelon conjugate with an appropriate solution of a radioactive metal and it is believed that the improved techniques for conjugation of proteins provided by the present invention will lead to improvements in radio-immunoassay technique, histo- and cytochemistry, and in vivo imaging. Once the protein-chelon conjugates have been prepared, they can be stored for long periods. It should subsequently be possible to label them whenever wished with alpha, beta, gamma, positron, and even neutron emitters under mild and strictly comparable conditions. The method will be equally applicable to NMR imaging with paramagnetic ions serving as contrast agents. If a gamma emitter is desired the protein may be labelled with 111In (specific activity  greater than 5000 Ci/mmol) while a suitable positron emitter is 68Ga (max. theoretical specific activity 2.7xc3x97107 Ci/mmol).
The protein or polypeptide derivatives of formula II which are used as starting materials in the coupling reaction of the present invention may be prepared by reacting a protein or polypeptide with a suitable bifunctional reagent using methods well known in the art. It is easily possible to, e.g. acylate side chain amino groups of proteins and polypeptides with bifunctional reagents. In that case when an aliphatic or aromatic compound of formula R3xe2x80x94Xxe2x80x94R1 is used containing one function (R3) capable of reacting with a reactive group of a protein side chain and a second reactive group R1, wherein R1 as well as X are as defined above, compounds of formula Axe2x80x94Xxe2x80x94R1 (II) are obtained wherein the protein or polypeptide residue is connected via a side chain. While in such a reaction, e.g., acylation will occur at several sites and a mixture of different compounds II will be obtained, it is preferable to use reaction conditions by which the point of attachment is limited to a single selected region of the protein or polypeptide. A preferred selected region in connection with the present invention is the carboxy terminus of the protein or polypeptide.
In recent years proteolytic enzymes have already been used in the synthesis of peptide bonds. This is possible because the enzyme catalyzed proteolysis is a reversible reaction. The method has been described by several authors (see e.g. Jakubke et al., Angew. Chemie, Int. Ed. Engl., 24, 85-93 [1985]) and has been used already successfully, e.g. in the preparation of human insulin (see e.g. Rose et al., Biochem. J. 211, 671-676 [1983]; European published patent application No. 87 238). Especially suitable enzymes useful in such reverse proteolysis which can be used to prepare compounds which are the preferred coupling reagents in the process of the present invention are trypsin and carboxypeptidase Y. However, other enzymes can also be used, with the best reaction conditions being easily determined in some preliminary experiments.
The general reaction can be described by the following equation 
wherein A, X and R1 are as defined above and the carboxyl group is the C-terminus of the molecule.
In specific examples the coupling of p-aminophenylalanine amide (with carboxypeptidase Y) and of m-amino-benzaldehyde methyl and ethyl acetal (with trypsin) to the C-terminal region of the B-chain of insulin is described below. These compounds are representations of bifunctional molecules of the general formula R3xe2x80x94Xxe2x80x94R1 which are especially useful in connection with the present invention. In these and all following reactions no protection whatever was needed for the protein""s functional groups. Under the semi-aqueous conditions that have been chosen for the trypsin-catalyzed reaction, synthesis is greatly favoured over hydrolysis. Only LysB29 is affected and the final product was des-AlaB30-insulin-B29-m-formylanilide.
Normally, carboxypeptidase Y progressively attacks the C-terminus of proteins. Such a degradation can be carried out under conditions that favour synthesis at the same time as hydrolysis (Widmer et al. in Peptides 1980, 46-55 (editor K. Brunfeldt). Scriptor Kopenhagen [1981]; Widmer et al. in Peptides 1982, 375-379 (eds. Blaha and Malon), W. de Gruyter Berlin and New York [1983]). Under such circumstances, while a mixture is obtained, it is one in which useful products predominate. If large polypeptides and proteins are used the heterogeneity of the end product which may exist due to continuous degradation of the protein from the C-terminus or perhaps of the enzyme""s inserting more than one molecule of compound R3xe2x80x94Xxe2x80x94R1, is, however, of minor importance since it remains restricted to a small region, viz. the C-terminus, of the molecule and will generally not be crucial to its activity.
The above coupling principle which can be extended to all proteins of interest is of specific interest in view of its applicability to immunoglobulins, especially antibodies of the IgG type, and to fragments thereof, such as F(abxe2x80x2)2 or Fab. Trypsin produces Fab-like fragments analogous to those made with papain, and the above equation applies to the fixation of a unique site for conjugation at the C-terminus thereof, a region known to be far from the antigen-binding site. It cannot be excluded that papain might also participate in such a coupling reaction and give directly the wanted derivatives of Fab fragments. Furthermore, carboxypeptidase Y will also introduce points of attachment (for the formation of Schiff base type, derivatives) at the C-termini of all the chains of IgG. F(abxe2x80x2)2, and Fab molecules, once again far from the antigen combining sites.
The feasibility of the carboxypeptidase Y approach has been studied in extenso. The possible range of conditions in the coupling of p-amino-phenylalanine amide to insulin with carboxypeptidase Y was explored in the following manner:
An aqueous solution of p-amino-phenylalanine amide (10 mg/ml) was adjusted to the desired pH in the range 5.5 to 9.5 with either dilute NaOH or dilute HCl as necessary, and then freeze dried. This product could then be dissolved in water to produce a self-buffered solution at any desired concentration in the range from 0.1 M to saturation. For each set of conditions 4.5 xcexcl of a solution of zinc-free insulin (20 mg/ml in 0.01 N HCl) was taken. 5.5 xcexcl of buffer (0.1 M sodium phosphate) were added at the desired pH. The p-amino-phenylalanine amide was then added in the form of 6.5 xcexcl of self-buffered solution at the appropriate pH and the chosen concentration. Carboxypeptidase Y (1 xcexcl of an aqueous solution of 2.14 mg protein/ml) was then added. If the solution was not clear (i.e. close to the isoelectric point of insulin), sufficient solid urea was added to clarify it. This system was used for the rapid exploration of a range of amide concentrations and reaction times. For each time point the degree of coupling and degradation was assessed in the first instance by quenching 2 xcexcl of the reaction mixture in 100 xcexcl of glacial acetic acid, diluting it to 1.7 ml in 0.01 N HCl, and applying 1 ml of the resulting solution for HPLC. Indications of success were confirmed by tests on the product isolated from larger scale digests after acid quenching by gel-filtration in 1% acetic acid. The reaction chosen was a Schiff base coupling with benzaldehyde and consecutive reduction with cyanoborohydride.
The best conditions found are: pH 8.5; a final concentration of p-amino-phenylalanine amide of 1.3 M; incubation from 7 to 22 hours at 20xc2x0 C. Digestion at pH values higher than pH 8.5 led to much slower reaction, whilst digestion at pH values lower than pH 8.5 led progressively to more degradation and less useful synthesis. At pH 5.5, or at practically any pH in the absence of p-amino-phenylalanine amide, the concentration of carboxypeptidase Y used in the above tests led to rapid and extensive degradation.
Additional experiments indicated that contrary to what is advantageous when using trypsin as enzyme the addition of butane-1,4-diol up to 50% by volume gives little advantage in carboxypeptidase-mediated couplings with the alpha amino group of p-amino-phenylalanine amide, but increases the coupling yield when the attacking nucleophile is a benzylamine derivative.
The feasibility of the carboxypeptidase Y approach having been demonstrated with insulin, conditions will have to be optimized for each new protein. With relatively few trial experiments, it will prove possible to find conditions that give useful yields of derivatives capable of coupling.
Once made in bulk, the butane-1,4-diol solutions can be stored for very long periods. Aminobenzaldehydes being well known for the spontaneous polymerization between their amino and aldehyde groups, had to be protected at the aldehyde function until the amino function was protected by combination with the protein. The acetal protection was sufficiently stable to survive all the steps of the synthesis, but the resulting protein aldehyde acetals are so labile to acid that they can be deprotected under conditions mild enough to present no risk to the integrity of most proteins.
Another preferred selected region for the introduction of a complementary group (amino or carbonyl function) is the amino terminus of the protein or polypeptide.
Thus an N-terminal glycyl residue of a protein may be converted into an aldehyde function, by transamination, preferably by reaction with glyoxylate. This reaction proceeds under relatively mild conditions (see e.g. Dixon, H. B. F. and Fields, R., Methods in Enzymology, 25, 409-419 [1979]). The generality of this reaction may be extended by deliberately introducing Gly as N-terminal residue of proteins produced by recombinant DNA methods. In cases where Gly is not N-terminal, a useful keto group may nonetheless be formed by transmination of another N-terminal amino acid than glycine to yield the corresponding keto acid.
Furthermore, N-terminal Ser and Thr residues may be oxidized in an exceedingly mild reaction with periodate (e.g. 20xc2x0 C. 26 xcexcM protein, 1 mM imidazole buffer pH 6.95, 2-fold excess of periodate for 5 min). N-terminal Ser reacts about 1000 times as fast as other protein groups (Fields, R. and Dixon. H. B. F., Biochem. J. 108, 883 [1968]), so great specificity may be obtained. For greater generality, N-terminal Ser or Thr may be introduced by recombinant DNA techniques, or, in appropriate cases, by selecting a source of the protein of interest which has a natural Ser or Thr N-terminus.
The polypeptidyl N-terminal aliphatic aldehydes produced by these techniques may be reacted, preferably, with aromatic amines or with O-alkyl-hydroxylamines.
This invention also encompasses intermediate compounds of the formula Axe2x80x94Xxe2x80x94R1xe2x80x2 (IIxe2x80x2) and R2xe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x2 (IIIxe2x80x2), in which A, X and Xxe2x80x2 are as defined above, and R1xe2x80x2 and R2xe2x80x2 are defined as R1 and R2 are defined above, except that they may additionally be protected amino groups. Bxe2x80x2 in formula IIIxe2x80x2 is a residue of DTPA, ferrioxamine B or desferrioxamine B, cuprioxamine B, polyglutamic acid and derivatives thereof or [Nxcex5 (DTPA-alanyl)-Lys]n, with n being an integer  greater than 1.
As discussed above (see page 10), preferred compounds of formula II are those wherein X is an aromatic or araliphatic radical or has O adjacent to the amino group where R1 is amino or protected amino. Another preferred group of compounds of formula II are derivatives of immunoglobulins, i.e. those wherein A represents the residue of an immunoglobulin molecule, preferably of an IgG or antibody molecule, or of a fragment thereof such as an Fab or F(abxe2x80x2)2 fragment. The preparation of the novel compounds can be effected according to methods well-known in the art, especially in the way described hereinbefore by reverse proteolysis.
The reaction partners of compounds II are compounds of formula R2xe2x80x94Xxe2x80x2xe2x80x94B (III) wherein R2, Xxe2x80x2 and B are as defined above. If two proteins or a protein and a polypeptide are to be linked together (formation of homo- and hetero-dimers) B is the residue of a protein or a polypeptide. The proteins or polypeptides are coupled via an amino or carbonyl function already present in the molecule or which is introduced by methods know in the art. A formyl function may be present in protected form as an acetal, preferably in form of a methyl or ethyl acetal.
The keto, aldehyde or acetalized aldehyde function in compounds II und III may be introduced either directly using reactions well known in the art or indirectly in the form of a non-carbonyl precursor group which can be converted into a carbonyl function by known methods, such as the periodate oxidation of a diol residue (see Examples 7 and 17) or of a residue with vicinal hydroxy and amino groups.
Regarding the reaction partners of compounds II, i.e. the compounds of formula III as defined above, those compounds are of most importance in connection with the present invention wherein B is the residue of a chelating agent (chelon). Any compound which is capable of chelating metal ions can be used. If the chelating agent does already contain a group R2, i.e. an amino or carbonyl function (in which case Xxe2x80x2 is absent), there is no need to introduce an additional functional group of that type and the chelon can be coupled directly to a compound of formula II above (unless it is wished to convert an aliphatic group R2 to a preferred aromatic group R2). 1-p-Aminophenylethylene-dinitrilo-tetraacetic acid (U.S. Pat. No. 3,994,966) is an example of such a compound which already contains an aromatic amino group. Other suitable chelating agents useful in the present invention and worth being mentioned are 1-amino-6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane, otherwise known as desferrioxamine or deferoxamine (in its iron-bound form known as ferrioxamine B) and diethylenetriaminepentaacetic acid (DTPA; Krejcarek et al., Biochem. Biophys. Res. Commun. 77, 581-585 (1977)). These latter two chelating agents may be converted into preferred derivatives R2xe2x80x94B by methods well known in the art.
Hitherto, desferrioxamine has been attached to proteins by means of random coupling to side-chain amino groups, brought about either by glutaraldehyde (Janoki et al., Int. J. Appl. Radiat. Isot. 34, 871-877 [1983]) or by water-soluble carbodiimide (Janoki et al., J. Nucl. Med. 24, 909 [1982]). These papers, while fully demonstrating the excellence of the choice of that chelon, also indicated the need to look for milder, more specific methods of coupling.
Ferrioxamine B is much more soluble than its iron-free form, desferrioxamine. Because it is more difficult to follow the syntheses with the iron-free compounds, since chromatography tends to be difficult, and there is also the danger that the hydroxylamino groups, if not protected by chelation, might participate in side reactions, ferrioxamine B instead of the iron-free compound was used to produce protein conjugates. Then the iron was removed with acid/EDTA and the metal-free form of the conjugate was stored until needed. This approach proved satifactory, as judged by the fact that the protein conjugate treated in this way could be loaded with 111In and 68Ga, whilst a protein conjugate that still had its iron could not be loaded. Therefore, ferrioxamine B by use of the following reaction sequence (Scheme 1) was transformed to m-aminobenzoyl-ferrioxamine B which is an example of a compound of general formula III and very useful as coupling partner in the process of preparing protein or polypeptide derivatives in accordance with the present invention. 
Instead of m-aminobenzoyl-ferrioxamine B there can also be used the analogous compound wherein the iron ion is replaced by Cu2+. This latter ion is sufficiently weakly bound to be replaced by other metals such as Fe3+ but otherwise strongly enough to remain in the complex during all the other operations described above.
The preparation of m-aminobenzoyl-ferrioxamine B and its coupling to an insulin derivate are described in detail in Examples 1(b) and (c) respectively (below). The copper analogues of these compounds can be made in an analogous manner to that described therein.
Cuprioxamine B is made precisely analogously to the published method for the iron complex (Prelog, V. and Walser, A., Helv. Chim. Acta 45, 631-637 [1962]) with an equivalent quantity of cupric chloride instead of ferric chloride used in that publication. The intermediate products, as well as the final m-aminobenzoyl-cuprioxamine B are all light green in colour. Their Rf values on thin-layer chromatography (t.l.c.) are all identical to those of the iron compounds.
Unlike in case of m-aminobenzoyl-ferrioxamine B, removal of the metal is not necessary before loading with another metal in the case of m-aminobenzoyl-cuprioxamine B. The binding constant of copper in the complex is many orders of magnitude lower than that for metals such as iron and gallium, which displace the copper almost instantly in dilute, neutral or mildly acid solution.
DTPA has usually been attached to proteins either by means of its bis-anhydride (e.g. Layne et al., J. Nucl. Med. 23, 627 [1982]), or by a mixed-anhydride method (Krejcarek and Tucker, Biochem. Biophys. Res. Comm. 77, 581 [1977)). In both cases, the coupling to the side chains of the target protein is random. In addition the bis-anhydride is capable of reacting with more than one amino group at a time, and does so to a considerable extent. This reagent is also very rapidly hydrolysed in aqueous media, and although this has not greatly hindered its exploitation so far, it could nonetheless be a complicating factor under some circumstances. Paik et al., J. Nucl. Med. 24, 932 (1983) formed a mixed anhydride between carefully controlled quantities of DTPA and isobutylchloroformate. However, as with the original work of Krejcarek and Tucker (supra) they were unable to avoid the formation of statistical mixtures of products, no matter what ratio of reactants was chosen. The mixed anhydride, too, was labile to water.
Therefore, in accordance with the present invention an activated derivative of DTPA was chosen that, because of its stability, could be purified from its bi-reactive form. This derivative is DTPA-mono-(m-formylanilide) (or its dimethyl or diethylacetal) the preparation of which is shown in the following scheme. 
While mention was made above of the desirability of keeping ferrioxamine B in its metal-bound form until the end of the synthesis of the protein-chelon conjugate, no such difficulty presents itself with the DTPA derivative. The synthesis can be carried out with metal-free compounds, and the final protein conjugate could be loaded with the desired ion without difficulty.
An example of very powerful compounds of formula III in terms of chelating activity, containing a polymeric chelon, ready to couple by the Schiff base method of the present invention are compounds of the formula m-NH2xe2x80x94C6H4xe2x80x94COxe2x80x94[Nxcex5-(DTPA-alanyl)-Lys]n, wherein n is are integer  greater than 1. The preparation of such a compound with n=5 and its coupling is described in detail in Example 6 below. Another compound of that type is, e.g., polyglutamic acid to which ferrioxamine B is coupled up to one ferrioxamine B per side chain carboxy group (see Example 15 below). However, in analogy to polyglutamic acid and derivatives thereof other polymeric compounds, especially polypeptides, may be used to form compounds of formula III of the present invention.
Finally, another monovalent derivative of the chelating group DTPA which can be used for labelling of polypeptides and proteins is DTPA-alanine-p-nitrophenylester, i.e. a compound of the following formula 
This compound can be prepared in the following manner:
To 2 ml of aqueous 1 M sodium acetate buffer, pH 5.5, were added 50 mg of Ala-p-nitrophenylester.HCl under vigorous vortex mixing at room temperature. As soon as the material had dissolved, 84 mg of DTPA-bisanhydride were added under further vigorous vortex mixing. The anhydride dissolved over a period of about two minutes. At this point the reaction mixture was injected onto a preparative HPLC column (250xc3x9716 mm, packed with 7 xcexcm LICHROSORB RP-8 particles) previously equilibrated with 0.1% (w/v) trifluoroacetic acid in water. The column was eluted at 2 ml/min with the same solvent for 5 min, whereupon a biphasic linear gradient of pure acetonitrile was applied, the first phase reaching 35% acetonitrile after 35 minutes and the second reaching 55% acetonitrile after 85 minutes total time. The eluent was held at 55% acetonitrile for 5 minutes before being programmed down linearly to 0% over 10 min. The effluent was monitored at 214 nm. The desired product was collected at retention time 57-62 min and the dimeric product, due to the acylation of two molecules of Ala-p-nitrophenyl ester by the bis-anhydride, eluted at retention time 74-95 min. After removal of acetonitrile at room temperature on a rotary evaporator, the product was recovered by lyophilisation (yield ca. 40 mg). The product was examined by FAB-MS and by analytical HPLC on a RADIALPAK xcexcBONDAPAK C-18 cartridge with a linear gradient of pure acetonitrile (0-60%, v/v, 2% per min.) after 5 minutes in 0.1% (v/v) aqueous CF3COOH. The desired product eluted at retention time 25 min under these conditions. Some batches which contained a contaminant, identified by FAB-MS as possessing an extra alanyl residue but only one nitrophenylester group and eluting from the analytical column at retention time 26 min were repurified on the preparative column (see Example 1(c)). Yield from 50 mg Ala-p-nitrophenylester.HCl: ca. 24 mg. The final product was pure by analytical HPLC and gave the expected FAB-MS spectrum (protonated molecular ion at m/z 586, sodium-cationised ion at m/z 608 and potassium-cationised ion at m/z 624).
The above mentioned compounds, wherein a chelating agent has been modified in order to make it suitable for a condensation reaction yielding Schiff bases, are still novel and, therefore, are also part of the present invention.